In previous studies, we introduced a doxycycline-inducible PAX3-FOXO1 expression construct and a constitutive MYCN expression construct into immortalized human myoblasts. PAX3-FOXO1 expression is up-regulated in these cells by doxycycline treatment and then down-regulated by doxycycline withdrawal. In cell culture studies, doxycycline treatment of these engineered myoblasts resulted in oncogenic transformation, as shown by focus formation assays. To study these events in vivo, mice were injected intramuscularly with these engineered cells, and progressively growing tumors (resembling human ARMS) formed when mice were fed a doxycycline-supplemented diet. To study the requirement for continued PAX3-FOXO1 expression, doxycycline was withdrawn after small palpable tumors formed, resulting in growth cessation, widespread differentiation, cell death and overall tumor regression to undetectable levels. These findings support the utility of this inducible system as a model of targeted therapy against the fusion protein. Following regression, tumors generally recurred several weeks later despite the continued absence of doxycycyline. Tthe fusion protein was not detectable in most recurrent tumors, though there was a smaller subset of cases in which PAX3-FOXO1 was re-expressed in a doxycycline-independent manner. Cell lines derived from recurrent tumors that did not express the fusion protein were transformed in culture and tumorigenic in vivo in the absence of doxycycline. Though primary tumor-derived lines were dependent on PAX3-FOXO1 and differentiated when doxycycline was removed, recurrent tumor-derived cell lines did not differentiate after doxycycline withdrawal and instead proliferated continuously. These combined findings are consistent with the occurence of additional oncogenic events in rare tumor cells, rendering these cells independent of the fusion protein and providing a mechanism for resistance to the model targeted therapy. To provide further evidence that many recurrent tumors do not require PAX3-FOXO1 expression, we used the CRISPR/Cas9 system to knock down PAX3-FOXO1 in the original engineered myoblasts and representative recurrent tumor-derived cells. Following transduction of a CRISPR construct targeting the PAX3 exon 5, PAX3-FOXO1 protein expression was knocked down to nearly undetectable levels in these lines in the presence of doxycycline. In oncogenicity assays, the original engineered cells transduced with the PAX3-specific CRISPR construct failed to form foci when treated with doxycycline, whereas control transduced cells formed numerous foci as expected. In contrast, the PAX3-specific CRISPR did not affect the ability of the postulated PAX3-FOXO1-independent recurrent tumor-derived lines to form foci in the absence or presence of doxycycline, confirming that the oncogenicity of these cells does not require fusion protein expression. However, in a recurrent tumor-derived line expressing PAX3-FOXO1 in a doxycycline-independent fashion, transforming activity (with or without doxycycline) was abolished by the PAX3-specific CRISPR construct, indicating that PAX3-FOXO1 expression is essential for transformation in this second category of recurrent tumors. To better understand the mechanistic features of this recurrence mechanism, we investigated whether PAX3-FOXO1-independent cells can arise in culture. The original engineered myoblasts were grown in various culture conditions in the presence of doxycycline, and then injected into mice without any further doxycycline treatment. Though cells grown under normal growth conditions did not produce tumors, a set of culture conditions was identified that often gives rise to doxycycline-independent tumors, thus recapitulating the recurrence process by an initial in vitro PAX3-FOXO1-dependent step. These culture conditions also resulted in rare cells that showed oncogenic behavior in culture in the absence of doxycycline and associated PAX3-FOXO1 expression. Therefore, in vitro conditions have been identified that may select or enrich for rare cells that are capable of forming PAX3-FOXO1-independent RMS tumors.